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CIN85 participates in Cbl-b-mediated down-regulation of receptor tyrosine kinases
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
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2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 42, 39666-39672 p.Article in journal (Refereed) Published
Abstract [en]

The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.

Place, publisher, year, edition, pages
2002. Vol. 277, no 42, 39666-39672 p.
Keyword [en]
3T3 Cells, Adaptor Proteins; Signal Transducing, Animals, CHO Cells, Carrier Proteins/*metabolism, Cell Line, Cell Line; Transformed, Cloning; Molecular, Cricetinae, Down-Regulation, Endocytosis, Epidermal Growth Factor/metabolism, Hela Cells, Humans, Ligands, Mice, Microscopy; Fluorescence, Models; Biological, Phosphoproteins/*metabolism, Phosphorylation, Platelet-Derived Growth Factor/metabolism, Precipitin Tests, Protein Binding, Protein Structure; Tertiary, Proto-Oncogene Proteins c-cbl, Proto-Oncogene Proteins c-kit/metabolism, Receptor Protein-Tyrosine Kinases/*metabolism, Time Factors, Transfection, Two-Hybrid System Techniques, Ubiquitin-Protein Ligases
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90465DOI: 10.1074/jbc.M205535200PubMedID: 12177062OAI: oai:DiVA.org:uu-90465DiVA: diva2:162828
Available from: 2003-04-28 Created: 2003-04-28 Last updated: 2013-06-12Bibliographically approved
In thesis
1. Cbl in Regulation of Growth Factor Receptor Endocytosis and Actin Dynamics
Open this publication in new window or tab >>Cbl in Regulation of Growth Factor Receptor Endocytosis and Actin Dynamics
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins belonging to the Cbl family are multidomain scaffolds that participate in numerous processes, assembling signaling complexes and mediating attachment of ubiquitin to receptor and non-receptor tyrosine kinases.

We characterized a novel role for Cbl and Cbl-b in ligand-dependent internalization of growth factor receptors. Upon stimulation with epidermal growth factor (EGF), Cbl proteins associate with EGF receptor, become phosphorylated, and bind to the three SH3 domains of CIN85, which brings endophilins to the complex with active receptors. Endophilins can induce internalization of the plasma membrane, contributing to formation of clathrin-coated pits. We identified a minimal binding domain for CIN85 in the carboxyl termini of Cbl/Cbl-b and observed constitutive association between CIN85, Cbl/Cbl-b and oncogenically stimulated receptor tyrosine kinases. In addition to functioning as a ubiquitin ligase, Cbl forms a complex with CIN85 and endophilin, which is required for efficient endocytosis and downregulation of membrane receptors.

In EGF stimulated cells, we observed inducible modification of CIN85 and related CMS proteins by attachment of a single ubiquitin molecule. Monoubiquitination of CIN85 was mediated by the RING finger and dependent on the carboxyl terminal part of Cbl/Cbl-b, and demanded an intact carboxyl terminus of CIN85. Prolonged stimulation with EGF induced concomitant degradation of EGF receptors, Cbl, and monoubiquitinated forms of CIN85 in lysosomes.

Cbl regulates cytoskeletal processes in a variety of cell systems. We identified SH3P2, a protein with SH3 domain and ankyrin repeats, as a Cbl partner and described its phosphorylation by Src and its distribution in fibroblasts and osteoclasts. SH3P2 formed inducible complexes with Cbl and actin in spread cells and colocalized with dynamic actin structures.

Our data contribute to better understanding of the role of Cbl in downregulation of receptor tyrosine kinases as well as in controlling actin rearrangement.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 58 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1243
Keyword
Cell and molecular biology, RTK, Cbl, endocytosis, ubiquitination, actin, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-3443 (URN)91-554-5583-2 (ISBN)
Public defence
2003-05-22, B42, Biomedical Center (BMC), Uppsala, 13:15
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Available from: 2003-04-28 Created: 2003-04-28 Last updated: 2013-06-10Bibliographically approved

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